DESCRIPTION: In humans and animals, the heme released from hemoglobin, myoglobin, and cytochrome P-450 degradation is converted to open-chain biliverdin by heme oxygenase (HO) catalysis. In these O2-dependent processes, carbon monoxide is released. HO activity found in brain tissue has implicated CO as a neurotransmitter. Nitric oxide synthase (NOS) also generates a diatomic gas molecule by catalyzing the conversion of L-arginine to citrulline, and the product NO is a major regulator in the nervous, immune, and cardiovascular systems. A target of this research project is the elucidation of the chemistry of O2- activation and the mechanism of catalysis of the heme cofactors in wild- type HO and NOS and in enzyme prepared with site-directed mutations that probe changes in heme structure and activity. The approaches of this project are to characterize oxygenated and oxidized intermediates of the heme cofactors by vibrational spectroscopy, coupled with a vigorous synthetic model approach using novel, highly protected metallo-porphyrins that even stabilize an Fe-O2 adduct at room temperature. Respiration- coupled energy transduction in all aerobic life forms is carried out by cytochrome oxidases. Cytochrome bd oxidase of E. coli is a terminal oxidase whose unusually high O2 affinity is associated with its d cofactor, a 5,6- dihydroxyprotochlorin. Research on this hydroporphyrin oxidase will be continued using the chromophore-specific technique of resonance Raman spectroscopy. A principal goal is the identification of the axial ligands of the cofactors. For chlorin d, this work seeks to confirm the proposal that this cofactor is unique in lacking a strong axial ligand. Work on the chemistry of the d cofactor will be supported by a new effective synthetic model. A third and expanding aspect of this research project is the investigation of the role of quinone cofactors in amine oxidase. As with the porhyrins, this work relies on accurate quinone model compounds for spectroscopic comparison. Crosslinking of connective tissue is carried out by copper lysil oxidase, an enzyme with a tyrosine-derived quinone at its active site. The proposed research will use Raman spectroscopy to identify the nature of quinone substituents in previously uncharacterized cofactors (as in lysil oxidase), in intermediates of the catalytic cycle, and during the biosynthesis of the cofactor.